CN106769969A - A kind of Sparklet testing method of antibody protein content - Google Patents

A kind of Sparklet testing method of antibody protein content Download PDF

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Publication number
CN106769969A
CN106769969A CN201710094368.2A CN201710094368A CN106769969A CN 106769969 A CN106769969 A CN 106769969A CN 201710094368 A CN201710094368 A CN 201710094368A CN 106769969 A CN106769969 A CN 106769969A
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antibody protein
absorbance
dilution
test sample
protein content
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CN106769969B (en
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黄峥
肖楠
庄超
郑琛
齐念民
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Shanghai Taiyin Biolog Technology Co Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultra-violet light

Abstract

The invention discloses a kind of Sparklet testing method of antibody protein content, comprise the following steps:(1) it is dilution gradient dilution test sample with denaturing liquid, after room temperature is placed, determines the absorbance of different extension rate solution;With the inverse of extension rate as abscissa, it is that ordinate draws standard curve to determine light absorption value.(2) extinction coefficient under the conditions of test sample non denatured is calculated, i.e., albumen is 1mg/mL under normal condition, light path is the absorbance of 1cm;(3) antibody protein concentration when light path is 1cm is calculated.Sample size is few needed for this method, routine need to using be milligram level antibody protein extinction coefficient is determined by amino acid composition analysis, again its concentration is determined with ultraviolet spectrometry absorption process, and this method can be used the antibody protein of Gamma Magnitude, only once experiment can be obtained by the extinction coefficient and respective concentration of the antibody protein, and standard items need not be used, it is simple to operate rapid, it is adaptable to be used during without protein standard substance.

Description

A kind of Sparklet testing method of antibody protein content
Technical field
The invention belongs to biological pharmacy technical field, it is related to antibody protein content detection technology, more particularly to a kind of antibody The Sparklet testing method of protein content.
Background technology
At present, the common method for determining protein content has:Kjeldahl's method, biuret method, Lowry methods, Bradford Method, ultraviolet absorption method etc..These methods respectively have quality, and different assay methods can be selected for different types of sample.
Kjeldahl's method is usually used in the measure of organic compound, and the reaction time is long, complex operation, big with sample amount.Biuret Method, Lowry methods and Bradford methods are the chromogenic assay method of protein solution and are both needed to standard items.Biuret method is usually used in It is quick but do not need exactly accurate measure, such as it is used for the measure of preceding several steps of protein purification.Lowry methods are chosen always It is the standard determination method of protein, this method is based in alkaline solution, the oxidizable composition (such as sulfydryl, phenolic group) of protein will Cu2+It is reduced to Cu+, Folin- phenol reagents quantitatively with Cu+Reaction, forms blue compound, and this blue compound has in 650nm Maximum absorption band.Recently, BCA methods newly developed are also the improvement on the basis of Lowry methods, simply BCA and Cu+Reaction ratio Folin- phenol reagents and Cu+Reaction it is stronger.The method reagent preparation is more complicated, cumbersome, and needs corresponding Standard items, general novel antibodies do not have corresponding standard items, therefore are not suitable for the detection of antibody protein content.Bradford methods Be called Coomassie Brilliant Blue, be dyestuff Coomassie brilliant G-250 in an acidic solution with the basic amino acid (arginine of protein Deng) and aromatic amino acid residue be combined, solution is changed into blue from brownish black, and maximum absorption wavelength is changed into 595nm, The absorbance determined under 595nm wavelength is linear with protein concentration.This method sensitivity is high, repeatability, but due to each Arginine is different with the content of aromatic amino acid in planting protein, and Bradford methods have larger when being determined for different proteins Deviation, therefore during usually using the method, need corresponding standard items, but be there is no statutory standards product generally for novel antibodies.
The detection method of conventional antibody protein content is mainly ultraviolet absorption method.The method requirement has disappearing for the albumen Backscatter extinction logarithmic ratio.Due to there is the tyrosine containing conjugated double bond, tryptophan, cystine etc. in protein, they have absorption purple The property of outer light, it absorbs peak at 280nm wavelength, and absorbance at this wavelength is proportional with its concentration, therefore The foundation that can be determined as quantification of protein.Common operation is:Made with the solvent (water or buffer solution) for preparing protein solution Blank is returned to zero, and protein solution is rule of thumb diluted to suitable concentration, make at its 280nm light absorption value 0.5~1.0 it Between, testing protein solution is poured into quartz colorimetric utensil, three parallel determinations, albumen is calculated according to Lambert-Beer laws The content of solution:
A=ε cl
In formula, A- absorbances, OD;ε-molar absorption coefficient, is once called as molar extinction coefficient;C- concentration, mol/L;L- colorimetrics Ware light path thickness, cm.
The minimum measurement volume of the method cuvette is 4uL, rule of thumb diluted protein solution, and different extension rates are calculated The concentration for obtaining is different, and accuracy is not high, and the molar extinction coefficient of unfolded protein can be subject to secondly level, the shadow of tertiary structure Ring, so as to change, can not simply be estimated.Generally determine that its extinction coefficient is constituted by determining amino acid Or calculated by model and tried to achieve.Protein content needed for two methods are a kind of reaches milligram level, it is a kind of theoretical calculate may result in compared with Big error, not as this law is measured accurately.
The content of the invention
The present invention provides a kind of Sparklet testing method of antibody protein content, more micro, accurately determine antibody protein The method of content, to solve the shortcoming and defect that existing A280 ultraviolet absorption methods determine protein concentration.Needed for the method for the present invention Sample size is few, simple to operate rapid, reproducible, and different types of antibody protein solution can be determined effectively.
The present invention is set up on the basis of existing A280 ultraviolet absorption methods, is denatured antibody protein completely with guanidine hydrochloride solution Unfolding, it is theoretical such that it is able to calculate it according to the quantity of tryptophan, tyrosine residue and disulfide bond etc. in test antibodies albumen Molar extinction coefficient.The present invention is tested using microcolorimetric ware, and linearity curve is made from antibody samples gradient dilution, is surveyed Amount volume is only 4 μ L, can calculate protein content result accurate with the relation between linear fit difference extension rate and absorbance Du Genggao.
Technical scheme is as follows:
A kind of Sparklet testing method of antibody protein content, comprises the following steps:
(1) test sample is carried out into gradient dilution by dilution of denaturing liquid, room temperature places a period of time, so that antibody egg It is denatured completely in vain, determines the absorbance of different extension rate solution;With the coefficient of dilution as abscissa, the absorbance for measuring is vertical Coordinate draws denaturation linearity curve, obtains denaturation linear equation Y=AX+B;
(2) according to the amino acid sequence of the test sample, sample is supplied in line computation using ExPASyProtParam tool Albumen is 1mg/mL under extinction coefficient under the conditions of product non denatured, i.e. normal condition, and light path is the absorbance of 1cm;
(3) described absorbance is substituted into denaturation linear equation Y=AX+B, calculates the complete albuminate for 1mg/mL When the coefficient of dilution (inverse of d);The antibody protein concentration of the test sample is calculated according to following formula:
C=1.0 × d × 1/L
In formula, c- antibody protein concentration, mg/mL;D- protein solution extension rates;L- is light path (microcolorimetric ware light Journey).
Preferably, another test sample is also carried out into same gradient dilution by dilution of non denatured liquid including step (4), so Other steps of repeat step (1), obtain non denatured linearity curve afterwards;Then by step (3), the complete albuminate is 1mg/ Coefficient of dilution during mL substitutes into non denatured linearity curve, obtains extinction coefficient during the test sample antibody protein undenatured i.e. normality ζ, the extinction coefficient is tried out in following equation, be directly used in the test sample antibody protein it is undenatured when concentration mensuration:
A=ζ * C*L
In formula, c- antibody protein concentration, mg/mL;A- absorbances, OD;ζ is extinction coefficient;L is light path, cm.
Preferably, the denaturing liquid described in step (1) is 6.3mM disodium hydrogen phosphate dodecahydrates, the hydration phosphorus of 13.7mM bis- Acid dihydride sodium, 6M guanidine hydrochlorides, the mixed liquor of pH=6.5;Non denatured liquid described in step (4) is the hypophosphite monohydrates of 6.3mM 12 Disodium hydrogen, the hypophosphite monohydrate sodium dihydrogens of 13.7mM bis-, the mixed liquor of pH=6.5 ± 0.1.
Preferably, the test sample described in step (1) and (4) is the antibody protein solution for obtaining after purification, and is free of There is the material for absorbing ultraviolet.
Preferably, make depending on the absorbance of test sample of the gradient dilution coefficient according in step (1) and (4) The absorbance that solution is determined after dilution is in the range of apparatus measures.
Preferably, the absorbance that solution is determined after dilution is between 0.056 to 0.42.
Preferably, the determining instrument of the absorbance described in step (1) and (4) is ultraviolet specrophotometer, such as Agilent Cary 60 etc..
Preferably, the parameter of described ultraviolet specrophotometer is set to wavelength 280nm, reading number of times three times, during operation Curvature correction is carried out, fit type is linear, minimum R2It is 0.99, measure cuvette is microcolorimetric ware, light path l is 0.1cm.
Preferably, before the absorbance of the test sample described in measure, returned to zero with described denaturing liquid, zeroing is inhaled Luminosity is below 0.37.
Preferably, according to extension rate sequentially determining from high to low, an extension rate solution has often been surveyed, it is clear with ultra-pure water After washing microcolorimetric ware, then survey next extension rate solution.
The invention also discloses Sparklet testing method the answering in antibody protein detection kit of above-mentioned antibody protein content With.
Compared with prior art, beneficial effects of the present invention are as follows:
Compared with existing A280 methods, the present invention has used micro ratio without determining extinction coefficient by other method A small amount of antibody samples after purification, so as to sample size needed for being greatly saved test, can be determined quickly by color ware, meet real Test room and Corporation R & D needs;Simultaneously not high in order to solve the problems, such as the existing method degree of accuracy, the present invention dilutes to protein solution Coefficient and absorbance carry out linear fit, it is to avoid different to protein solution extension rate, and causes the test sample egg for calculating Bai Hanliang different problems;And the method for the present invention is reproducible, Method validation precision RSD is respectively less than 5%;With can basis The design feature of different antibody proteins changes molar extinction coefficient, it is adaptable to the measure of various antibody protein contents, using model Enclose wide.
Relation between method of the present invention linear fit difference extension rate and antibody protein solution absorbance, solves Original A280 ultraviolet absorption methods accuracy problem not high, the protein content to different antibodies sample can be determined effectively.
Brief description of the drawings
Fig. 1 be the denaturing liquid gradient dilution of the present embodiment 1 after antibody 1 measure with extension rate inverse as X-axis-with inhale Luminosity is the denaturation linear diagram of Y-axis;
Fig. 2 be the non denatured liquid gradient dilution of the present embodiment 1 after antibody 1 measure with extension rate inverse as X-axis-with Absorbance is the non denatured linear diagram of Y-axis;
Fig. 3 be the denaturing liquid gradient dilution of the present embodiment 2 after antibody 2 measure with extension rate inverse as X-axis-with inhale Luminosity is the denaturation linear diagram of Y-axis;
Fig. 4 be the non denatured liquid gradient dilution of the present embodiment 2 after antibody 2 measure with extension rate inverse as X-axis-with Absorbance is the non denatured linear diagram of Y-axis.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this hair It is bright, rather than limit protection scope of the present invention.Those skilled in the art are according to changing that the present invention makes in actual applications Enter and adjust, still fall within protection scope of the present invention.
When the present invention does not do special explanation, technological means used is ripe for those skilled in the art in following examples The conventional technical means known, and raw material, reagent used is also commercially available commercially available commodity.
Embodiment 1 determines the antibody protein content of antibody 1
(1) it is denatured the making of linearity curve:With denaturing liquid as dilution by 10 times of 1 gradient dilution of antibody, 20 times, 40 times, 80 times, 160 times, 320 times, after room temperature is placed 1 hour, determine the absorbance of different extension rate solution;It is (dilute with the coefficient of dilution Release the inverse of multiple) it is abscissa, the light absorption value of measure is drawn for ordinate and is denatured linearity curve, as shown in Figure 1.
Described denaturing liquid is 6.3mM disodium hydrogen phosphate dodecahydrates, the hypophosphite monohydrate sodium dihydrogens of 13.7mM bis-, 6M hydrochloric acid Guanidine, the mixed liquor of pH=6.5;Described antibody 1 is upper Haitai because of Bioisystech Co., Ltd's fermentation, purifying gained;Dilution institute Solution mensuration absorbance value between 0.056 to 0.42.
(2) protein structure according to antibody 1 is amino acid sequence, using ExPASyProtParam tool in line computation Its theoretic extension coefficient.
(3) absorptivity for obtaining step (2), the i.e. absorbance 1.304 of the complete albuminates of 1mg/mL substitutes into institute Obtain mark song:Y=4.1728x-0.007, extension rate when obtaining protein content for 1mg/mL is 3.18, substitutes into C=1.0 × d × 1/L calculating antibody protein concentrations:C=1.0*3.18*1/0.1=31.8mg/mL.(4) non denatured linearity curve is drawn, is passed through Extinction coefficient when calculating antibody 1 is undenatured, to the denaturation linearity curve of rectification step (1), and can be directly used for confession examination Concentration mensuration when sample antibody albumen is undenatured:With non denatured liquid as dilution by 10 times of 1 gradient dilution of antibody, 20 times, 40 Again, 80 times, 160 times, 320 times, the absorbance of different coefficient of dilution solution is determined;With the coefficient of dilution (i.e. extension rate fall Number) it is abscissa, it is that ordinate draws non denatured linearity curve to determine light absorption value, as shown in Figure 2.
Described non denatured liquid is 6.3mM disodium hydrogen phosphate dodecahydrates, the hypophosphite monohydrate sodium dihydrogens of 13.7mM bis-, pH6.5 ± 0.1 mixed liquor;Described antibody 1 is upper Haitai because of Bioisystech Co., Ltd's fermentation, purifying gained;Dilution resulting solution Mensuration absorbance value is between 0.056 to 0.42.
Coefficient of dilution during by step (3) gained protein content for 1mg/mL substitutes into non denatured normal linearity curve, calculates Obtain the antibody 1 it is undenatured when extinction coefficient be:ε '=1.37.
Embodiment 2 determines the antibody protein content of antibody 2
(1) it is denatured the making of linearity curve:With denaturing liquid as dilution by 3.33 times of 2 gradient dilution of antibody, 6.66 times, 8.88 times, 13.32 times, 17.76 times, after room temperature is placed 1 hour, determine the absorbance of different extension rate solution;To dilute Coefficient (i.e. the inverse of extension rate) is abscissa, and mensuration absorbance value is that ordinate draws denaturation linearity curve, such as Fig. 3 institutes Show.
Described denaturing liquid is 6.3mM disodium hydrogen phosphate dodecahydrates, the hypophosphite monohydrate sodium dihydrogens of 13.7mM bis-, 6M hydrochloric acid Guanidine, the mixed liquor of pH=6.5;Described antibody 2 is upper Haitai because of Bioisystech Co., Ltd's fermentation, purifying gained;Dilution institute Between solution mensuration absorbance value 0.056 to 0.42.
(2) according to the protein structure of antibody 2, using ExPASyProtParam tool in its extinction coefficient of line computation:
(3) by the absorptivity obtained by step (2), i.e. the absorbance 1.567 of the complete albuminates of 1mg/mL substitutes into institute Obtain mark song:Y=1.6003x-0.0228, extension rate when obtaining protein content for 1mg/mL is 1.01, substitutes into C=1.0 × d × 1/L calculating antibody protein concentrations:C=1.0*1.01*1/0.1=10.1mg/mL.
(4) non denatured linearity curve is drawn, by extinction coefficient of the calculating antibody 2 when undenatured, to rectification step (1) Denaturation linearity curve, and can be directly used for the test sample antibody protein it is undenatured when concentration mensuration:It is with non denatured liquid 3.33 times, 6.66 times, 8.88 times, 13.32 times, 17.76 times of 2 gradient dilution of antibody is determined different extension rates molten by dilution The absorbance of liquid.With the coefficient of dilution (i.e. the inverse of extension rate) as abscissa, mensuration absorbance value is that ordinate draws mark Directrix curve, as shown in Figure 4.
Described non denatured liquid is 6.3mM disodium hydrogen phosphate dodecahydrates, the hypophosphite monohydrate sodium dihydrogens of 13.7mM bis-, pH6.5 ± 0.1 mixed liquor;Described antibody 2 is upper Haitai because of Bioisystech Co., Ltd's fermentation, purifying gained;3.33 times of institutes of dilution Obtain solution mensuration absorbance value and be less than 1.
The coefficient of dilution (inverse of extension rate) during by step (3) gained protein content for 1mg/mL substitutes into non denatured Linearity curve, calculate the antibody 2 it is undenatured when extinction coefficient be:ε '=1.57.
The checking of the method precision of embodiment 3
3.1 instruments repeatability:Same date same analysis personnel make one group of mark song solution to test sample, to this group mark Bent solution repeats sample introduction and determines 6 times, obtains the standard curve of 6 times, and test sample protein concentration is calculated respectively.The results are shown in Table 1,6 Secondary calculating gained test sample protein concentration RSD is 1.30%.
The instrument repeated experiment result of table 1
Sample introduction sequence number Concentration (mg/mL)
1 28.36
2 27.95
3 28.29
4 28.92
5 28.32
6 28.85
Average value 28.45
Standard deviation 0.37
Relative standard deviation % 1.30%
3.2 sample repeatabilities:Same date same analysis personnel make two groups of solution to same test sample, to this two groups Solution repeats sample introduction 3 times respectively, and 6 sublinear curves are obtained, and test sample protein concentration is calculated respectively.The results are shown in Table 2,6 times It is 3.99% to calculate gained test sample protein concentration RSD.
The sample repeatability experimental result of table 2
3.3 Intermediate precisions:Different analysis personnel make solution to same test sample in not same date, and sample introduction is determined, 3 sublinear curves are obtained, test sample protein concentration is calculated respectively.The results are shown in Table 3,3 gained test sample albumen dense Degree RSD is 3.89%.
The Intermediate precision experimental result of table 3
The usage amount Gamma Magnitude of this method is just much of that, and this method need not use standard items, uses microcolorimetric ware, behaviour Make simple rapid, instead of conventional method is that two kinds of experiments are combined the tedious steps that can just obtain a result, the method for the present invention Relation between linear fit difference extension rate and antibody protein solution absorbance, solves original A280 ultraviolet absorption methods essence Exactness problem not high, the protein content to different antibodies sample can be determined effectively.
The invention also discloses a kind of kit, including denaturing liquid and non denatured liquid, the kit is used for antibody protein The trace detection of content.
Present invention disclosed above preferred embodiment is only intended to help and illustrates the present invention.Preferred embodiment is not detailed All of details is described, it is only described specific embodiment that the invention is not limited yet.Obviously, according to the content of this specification, Can make many modifications and variations.This specification is chosen and specifically describes these embodiments, is to preferably explain the present invention Principle and practical application so that skilled artisan can be best understood by and utilize the present invention.The present invention is only Limited by claims and its four corner and equivalent.

Claims (10)

1. a kind of Sparklet testing method of antibody protein content, it is characterised in that comprise the following steps:
(1) test sample is carried out into gradient dilution by dilution of denaturing liquid, room temperature places a period of time, determines different dilutions times The absorbance of number solution;With the coefficient of dilution as abscissa, the absorbance for measuring is that ordinate draws denaturation linearity curve, is obtained Denaturation linear equation Y=AX+B;
(2) it is non-in line computation test sample using ExPASyProtParam tool according to the amino acid sequence of the test sample Albumen is 1mg/mL under extinction coefficient under Denaturing, i.e. normal condition, and light path is the absorbance of 1cm;
(3) described absorbance is substituted into denaturation linear equation Y=AX+B, when calculating the complete albuminate for 1mg/mL The coefficient of dilution;The antibody protein concentration of the test sample is calculated according to following formula:
C=1.0 × d × 1/L
In formula, c- antibody protein concentration, mg/mL;D- protein solution extension rates;L- is light path.
2. the Sparklet testing method of a kind of antibody protein content according to claim 1, it is characterised in that also including step (4) another test sample is carried out into same gradient dilution by dilution of non denatured liquid, then other steps of repeat step (1), Obtain non denatured linearity curve;Coefficient of dilution during then by step (3) the complete albuminate for 1mg/mL substitutes into non denatured Linearity curve, obtains extinction coefficient ζ during the test sample antibody protein undenatured i.e. normality, and the extinction coefficient is tried out in following public affairs Formula:
A=ζ * C*L
In formula, c- antibody protein concentration, mg/mL;A- absorbances, OD;ζ is extinction coefficient;L is light path, cm.
3. the Sparklet testing method of a kind of antibody protein content according to claim 2, it is characterised in that in step (1) Described denaturing liquid is 6.3mM disodium hydrogen phosphate dodecahydrates, the hypophosphite monohydrate sodium dihydrogens of 13.7mM bis-, 6M guanidine hydrochlorides, pH=6.5 Mixed liquor;Non denatured liquid described in step (4) is 6.3mM disodium hydrogen phosphate dodecahydrates, the hypophosphite monohydrates two of 13.7mM bis- Hydrogen sodium, the mixed liquor of pH=6.5 ± 0.1.
4. the Sparklet testing method of a kind of antibody protein content according to claim 2, it is characterised in that step (1) and (4) test sample described in is the antibody protein solution for obtaining after purification, and does not contain the material for absorbing ultraviolet.
5. the Sparklet testing method of a kind of antibody protein content according to claim 2, it is characterised in that step (1) and (4) in depending on the absorbance of test sample of the gradient dilution coefficient according to, the absorbance of solution measure after dilution is made In the range of apparatus measures.
6. a kind of Sparklet testing method of antibody protein content according to claim 5, it is characterised in that solution after dilution The absorbance of measure is between 0.056 to 0.42.
7. the Sparklet testing method of a kind of antibody protein content according to claim 2, it is characterised in that step (1) and (4) determining instrument of the absorbance described in is ultraviolet specrophotometer, the parameter setting of described ultraviolet specrophotometer It is wavelength 280nm, reading number of times three times carries out curvature correction during operation, fit type is linear, minimum R2It is 0.99, determines ratio Color ware is microcolorimetric ware, and light path l is 0.1cm.
8. the Sparklet testing method of a kind of antibody protein content according to claim 7, it is characterised in that described determining Test sample absorbance before, returned to zero with described denaturing liquid, zeroing absorbance below 0.37.
9. the Sparklet testing method of a kind of antibody protein content according to claim 2, it is characterised in that according to dilution times Sequentially determining from high to low is counted, an extension rate solution has often been surveyed, after cleaning microcolorimetric ware with ultra-pure water, then surveys next Extension rate solution.
10. the Sparklet testing method of the antibody protein content described in claim 1~9 protein content detection kit should With.
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